Figure 4.
Removal of AP-1/Ent5 causes Kex2 to enter secretory vesicles.(A) Maturation kinetics of Kex2 compared with Sec7. A strain expressing Sec7-mScarlet and the processing protease Kex2-GFP was grown to mid-log phase and imaged by 4D confocal microscopy. Shown are average projected Z-stacks at the indicated time points from part 1 of Video 4. The upper row shows the complete projections, the second row shows edited projections that include only the cisterna being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Scale bar, 2 µm. (B) Quantification of tagged Golgi proteins during a typical maturation event. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in A. (C) Smoothed and averaged traces showing the relative kinetic signatures of Kex2 and Sec7. Data were obtained for 10 representative cisternae. (D) Maturation kinetics of Kex2 compared with Sec7 in an apl4Δ ent5Δ strain. The experiment was performed as in A. Shown are average projected Z-stacks at the indicated time points from part 2 of Video 4. Scale bar, 2 µm. (E) Quantification of tagged Golgi proteins during a typical maturation event in an apl4Δ ent5Δ strain. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in D. (F) Smoothed and averaged traces showing the relative kinetic signatures of Kex2 and Sec7 in an apl4Δ ent5Δ strain. Data were obtained for 10 representative cisternae. (G) Localization of Kex2 to sites of polarized secretion in apl4Δ ent5Δ cells after CK-666 treatment. APL4 ENT5 or apl4Δ ent5Δ cells expressing Sec7-mScarlet, Sec2-GFP, and Kex2-HaloTag were grown to mid-log phase, labeled with JFX dye, and imaged by confocal microscopy 15 min after treatment with CK-666. Shown are average projected Z-stacks. Individual fluorescence channels are shown in grayscale with merged images on the right. The pair of budded cells of the APL4 ENT5 strain is typical for the population, while the pair of budded cells of the apl4Δ ent5Δ strain is a striking example that illustrates the population trend. Scale bar, 2 µm. Quantification of the projected images was performed with the 60–75% of the cells in which Sec2 was concentrated at the bud neck or in a small or nascent bud. Approximately 10–20% of those cells were excluded from further consideration because they showed Sec7-labeled TGN structures in close proximity to Sec2. For the APL4 ENT5 strain, 109 cells were deemed suitable for analysis, and Kex2 overlapped with Sec2 in none (0%) of those cells. For the apl4Δ ent5Δ strain, 153 cells were deemed suitable for analysis, and Kex2 overlapped with Sec2 in 145 (95%) of those cells.

Removal of AP-1/Ent5 causes Kex2 to enter secretory vesicles. (A) Maturation kinetics of Kex2 compared with Sec7. A strain expressing Sec7-mScarlet and the processing protease Kex2-GFP was grown to mid-log phase and imaged by 4D confocal microscopy. Shown are average projected Z-stacks at the indicated time points from part 1 of Video 4. The upper row shows the complete projections, the second row shows edited projections that include only the cisterna being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Scale bar, 2 µm. (B) Quantification of tagged Golgi proteins during a typical maturation event. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in A. (C) Smoothed and averaged traces showing the relative kinetic signatures of Kex2 and Sec7. Data were obtained for 10 representative cisternae. (D) Maturation kinetics of Kex2 compared with Sec7 in an apl4Δ ent5Δ strain. The experiment was performed as in A. Shown are average projected Z-stacks at the indicated time points from part 2 of Video 4. Scale bar, 2 µm. (E) Quantification of tagged Golgi proteins during a typical maturation event in an apl4Δ ent5Δ strain. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in D. (F) Smoothed and averaged traces showing the relative kinetic signatures of Kex2 and Sec7 in an apl4Δ ent5Δ strain. Data were obtained for 10 representative cisternae. (G) Localization of Kex2 to sites of polarized secretion in apl4Δ ent5Δ cells after CK-666 treatment. APL4 ENT5 or apl4Δ ent5Δ cells expressing Sec7-mScarlet, Sec2-GFP, and Kex2-HaloTag were grown to mid-log phase, labeled with JFX dye, and imaged by confocal microscopy 15 min after treatment with CK-666. Shown are average projected Z-stacks. Individual fluorescence channels are shown in grayscale with merged images on the right. The pair of budded cells of the APL4 ENT5 strain is typical for the population, while the pair of budded cells of the apl4Δ ent5Δ strain is a striking example that illustrates the population trend. Scale bar, 2 µm. Quantification of the projected images was performed with the 60–75% of the cells in which Sec2 was concentrated at the bud neck or in a small or nascent bud. Approximately 10–20% of those cells were excluded from further consideration because they showed Sec7-labeled TGN structures in close proximity to Sec2. For the APL4 ENT5 strain, 109 cells were deemed suitable for analysis, and Kex2 overlapped with Sec2 in none (0%) of those cells. For the apl4Δ ent5Δ strain, 153 cells were deemed suitable for analysis, and Kex2 overlapped with Sec2 in 145 (95%) of those cells.

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